The present invention lies in the field of serine proteases, in particular serine proteases cleaving a post proline bond, such as the exopeptidase dipeptidyl peptidase IV (DPPIV) and the endopeptidase prolyl oligopeptidase (PO or PEP).
The invention relates to synthetic inhibitors of PEP and dipeptidyl peptidase IV, methods of their preparation and their therapeutic and diagnostic use.
Many biologically important peptide sequences contain proline. It confers unique conformational constraints on the peptide chain because the .alpha.-nitrogen atom of proline is part of the rigid pyrrolidine ring and, at the same time, is covalently bound by means of a secondary amide bond to the N-terminal preceding amino acid. Thus, the side-chain is cyclized back on to the backbone amide position. Inside an .alpha.-helix the possibility of making hydrogen bonds to the preceding turn is thereby lost and a kink will be introduced in the helix. The conformational restrictions imposed by proline motifs in a peptide chain probably implicate important structural or biological functions since a high degree of conservation is bound in many proteins and peptides.
An endo or C-terminal Pro-Pro bond and an endo pre-Pro peptide bond possess a high degree of resistance to any mammalian proteolytic enzyme. Only a limited number of peptidases are known to be able to hydrolyse proline adjacent bonds. Their activity is restricted by the isomeric state and the position of proline in the peptide chain. Dipeptidyl peptidase IV (DPPIV, also commonly referred to as CD26) is one of these peptidases. It is a serine protease which specifically cleaves N-terminal dipeptides from proteins and polypeptides carrying an unsubstituted N-terminus and a penultimate proline (or alanine) residue in trans conformation. DPPIV is a homodimer with subunit molecular weight of 110 kDa. It is a membrane protein expressed on the surface of lymphoid cells, epithelial and endothelial cells. Surface expression is increased significantly by T-cell activation. DPPIV has been reported to play a role in numerous processes such as T-cell costimulation, binding to proteins of the extracellular matrix, attachment of cancer cells to endothelium, binding to plasma adenosine deaminase, cooperation with entry of human immunodeficiency virus (HIV) into lymphoid cells.
The proteolytic activity of DPPIV resides in a stretch of approximately 200 amino acids located at the C-terminal end of the protein. The catalytic residues (Ser-629, Asp-708, His-740) are arranged in a unique order which is different from the classical serine proteases such as chymotrypsin and subtilisin. Proline specific dipeptidyl peptidase activity alters the biological activity of a large number of bioactive proteins and polypeptides comprising, amongst others, the neurotransmitter substance P, human growth hormone-releasing factor, erythropoietin, interleukin 2 and many others. Since many of these peptides have important effector functions, abnormal DPPIV activity--either too low or too high--will be reflected in an abnormal biological effect. Potential DPPIV substrates are listed in tables 1 and 2. The skilled person will easily recognize that interference with the effector function of these polypeptides may result in clinical conditions including inflammation, vascular diseases, auto-immune disease, multiple sclerosis, joint diseases and diseases associated with benign and malign cell transformation.
TABLE 1 ______________________________________ Human cytokines, growth factors, neuro- and vasoactive peptides with a penultimate proline, which are putative substrates for DPP IV Polypeptide N-terminal sequence ______________________________________ Interleukin-1.beta. Ala-Pro-Val-Arg-Ser- Interleukin-2 Ala-Pro-Thr-Ser-Ser- Interleukin-5 Ile-Pro-Thr-Glu-Ile- Interleukin-6 Val-Pro-Pro-Gly-Glu- Interleukin-10 Ser-Pro-Gly-Gln-Gly- Interleukin-13 (recombinant) Ser-Pro-Gly-Pro-Val- Complement C4a Lys-Pro-Arg-Leu-Leu- Granulocyte chemotactic protein II Gly-Pro-Val-Ser-Ala- Granulocyte macrophage colony stimulating Ala-Pro-Ala-Arg-Ser- factor Granulocyte colony stimulating factor Thr-Pro-Leu-Gly-Pro Erythopoietin Ala-Pro-Pro-Arg-Leu- Gastrin releasing peptide Growth hormone Phe-Pro-Thr-Ile-Pro- Interferon inducible peptide 10 (.gamma.IP10) Val-Pro-Leu-Ser-Arg- Interferon regulatory factor 1 (IRF-1) Interferon regulatory factor 2 (IRF-2) Insulin-like growth factor-1 Gly-Pro-Glu-Thr-Leu- Melanoma growth stimulating activity Ala-Pro-Leu-Ala-Thr- Migration inhibition factor Met-Pro-Met-Phe-Ile- Monocyte chemotactic protein I Glu-Pro-Asp-Ala-Ile- Neuropeptide Y Tyr-Pro-Ser-Lys-Pro- Pancreatic polypeptide Ala-Pro-Leu-Glu-Pro- Peptide YY Try-Pro-Ile-Lys-Pro- Prolactin Leu-Pro-Ile-Cys-Pro- RANTES Ser-Pro-Tyr-Ser-Ser- Substance P* Arg-Pro-Lys-Pro-Gln- Thrombopoietin Ser-Pro-Ala-Pro-Pro- Transforming protein (N-myc) version 1 Met-Pro-Gly-Met-Ile- Transforming protein (N-myc) version 2 Met-Pro-Ser-Cys-Ser- Tumor necrosis factor .beta. Leu-Pro-Gly-Val-Leu- Vascular endothelial growth factor Ala-Pro-Met-Ala-Glu- ______________________________________ *hydrolysis by DPP IV has already been proven (4, 5).
TABLE 2 ______________________________________ Human peptides and proteins with a penultimate alanine, which are putative substrates for DPP IV Polypeptide ______________________________________ adenosine deaminase annexins breast basic conserved protein cofilin natural killer cell enhancing factor b precursors of interferon .alpha. precursors of interleukin 1-.alpha. and .beta., and interleukin 13 precursors of macrophage inflammatory protein-2-.alpha. and .beta. precursor of melanocyte stimulating hormone precursor of oxytocin-neurophysin 1 Growth hormone releasing hormone* .beta. amyloid protein (1-28) Anxiety peptide `joining peptide` of pro-opiomelanocortin ______________________________________ *hydrolysis by DPP IV has already been proven (6).
DPPIV activity in serum is decreased in patients with auto-immune diseases and in patients receiving immunosuppressant drugs (Hagihara, M., Ohhashi, M., Nagatsu, T. (1987) Clin. Chem. 33, 1463-1465; Doctoral thesis I. De Meester, 1992, "Characterization of Human Lymphocytic Dipeptidyl Peptidase IV and its identification as the Activation Antigen CD26", Universitaire Instelling Antwerpen, Departement Farmaceutische Wetenschappen.
Examples of auto-immune diseases are rheumatoid arthritis, systemic lupus erythematosus (this is a vascular disease), multiple sclerosis, Serum DPPIV activity is also decreased in malignancies such as gastrointestinal cancers (Haacke, W., Kullerts, G., & Barth, A. (1986) Arch. Geschwulstforsch 56, 145-153) and blood cancers including acute lymphoid leukemia, lymphosarcoma and Hodgkin's disease (Kojima, J., Ueno, Y., Kasugai, H., Okuda, S., & Akedo, H. (1987) Clin. Chim. Acta 167, 285-291; Fujita, K., Hirano, M., Tokunaga, K., Nagatsu, T., & Sakakibara, S. (1977&gt;) Clin. Chim. Acta 81, 215-217). DPPTV activity is increased in patients with hepatobiliary disease and hepatic cancer (Burchardt, U., Klagge, M., Hafstein, M., Salka, S., Graupner, H., Lenski, K., Delev, B., Neubert, K. & Barth, A. (1987) Z. Klin. Med. 42, 245-249). A role in inflammation can also be inferred from the observation that DPPIV is involved in costimulation and proliferation of granulocytes and macrophages (Bristol, L. A., Sakaguchi, K., Appella, E., Doyle, D., & Takacs, L. (1992) J. Immunol. 149, 367-372).
Also the response of T-lymphocytes to recall antigen and the entry of HIV have been reported to be at least partially influenced by the catalytic site of DPPIV. Recall antigen can be defined as an antigen that activates the immunological memory. It is an antigen to which the individual has been exposed before and which has previously triggered an immunological response. Tanaka et al. (Proc. Natl. Acad. Sci. USA 90, 4586-4590 (1993)) demonstrated that the active site of DPPIV is involved in the response to recall antigen. Callebaut et al. (Science 262, 2045-2046 (1993)) found an effect of catalytic site ligands in their model of HIV entry.
Prolyl endopeptidase (PEP), also called proline oligopeptidase (PO), was first discovered by Walter and coworkers as an oxytocin-degrading enzyme in the human uterus (Walter et al., Science 173, 827-829 (1971)). The enzyme cleaves peptide bonds at the carboxy-side of proline in peptides containing the sequence X-Pro-Y, where X is a peptide or N-terminal substituted amino-acid and Y is a peptide, amino acid, amide or alcohol (Yoshimoto et al., J. Biol. Chem. 253, 3708-3716 (1979)). The enzyme has a high specificity for the trans-conformation of the peptide bond at the imino-side of proline (Lin & Brandts, Biochemistry 22, 4480-4485 (1983)).
Prolyl endopeptidase has been characterized and purified form plants, micro-organisms and various mammalian sources. The enzyme is widely distributed among different mammalian tissues and high activity is found in brain, kidney, lung and muscle. The human enzyme has been purified from lung, brain, erythrocytes and placenta (Kalwant & Porter, Biochem. J. 276, 237-244 (1992)).
The sequences of prolyl oligopeptidase from A. hydrophila (Kanatani et al., J. Biochem. 113, 790-796 (1993)), F. meningosepticum (Yoshimoto et al., Agric. Biol. Chem. 55, 37-43 (1991)), porcine brain (Rennex et al., Biochemistry 30, 2195-2203 (1991)) and human lymphocytes (Vanhoof et al., Gene 149, 363-366 (1994)) have been determined.
In vitro PEP catalyzes the cleavage of several biologically active peptides such as angiotensin II, oxytocin, vasopressin, bradykinin. In vivo however its function remains obscure (Welches et al., Life Sci. 52, 1461-1480 (1993)). Recently it was shown that the prolyl oligopeptidase activity in plasma correlates well with the different stages of depression. Maes et al. (Biological Psychiatry 30, 577-586 (1991)) showed that DPPIV is decreased in major depression. It was proven to be the best biochemical marker for depression found so far.
Hydrolysis of angiotensin I and angiotensin II by prolyl oligopeptidase results in the liberation of angiotensin (1-7). Angiotensin (1-7) has vasodilator activity and modulates the release of vasopressin, which is able to influence the process of memory as was shown by injecting rats with specific PEP-inhibitors. The injection reverses the scopolamine induced amnesia. This experiment is not only an example which provides evidence for a possible physiologic function for the enzyme, but moreover it has led to the hypothesis that inhibitors for PEP can influence the memory process and counter dementia (Yoshimoto et al., J. Pharmacobio-Dyn. 10, 730-735 (1987); Furukawa et al., J. Pharmacobio-Dyn 12, S136 (1989)).
PEP may also be involved in the regulation of the blood pressure by participating in the renin-angiotensin system. PEP can decrease the amount of angiotensin II and its precursor and moreover the peptide generated by cleavage by PEP, angiotensin (1-7) itself has vasodilator activity (Kohara et al., Hypertension 17, 131-138 (1991)). Furthermore, involvement of PEP in vascular disease can be inferred from its ability to cleave bradykinin (which controls blood pressure) and blood coagulation factor X.
As indicated above, the proline specific serine proteases probably have a role in various physiological processes in the human and animal body, which processes might be related to various disease states, both physical and mental.
It is an object of the invention to interfere in the physiological action of proline specific serine proteases, such as PEP and DPPIV, to be able to treat the said disease states.